Novel therapeutic indication of azithromycin for treatment of non-infective inflamatory diseases

ABSTRACT

The invention relates to the use of 9-deoxo-9-dihydro-9 a -methyl-9 a -homoerythromycin. A (generic name: azithromycin) for the therapy of neutrophil-dominated non-infective inflammatory diseases, pharmaceutical compositions containing azithromycin for enteral or parenteral administration and methods for the production of these pharmaceutical compositions.

The invention relates to the use of9-deoxo-9-dihydro-9a-methyl-9a-aza-9a-homoerythromycin A (generic name:azithromycin) for the therapy of neutrophil-dominated non-infectiveinflammatory diseases, pharmaceutical compositions containingazithromycin for enteral or parenteral administration and methods forthe production of these pharmaceutical compositions.

Most inflammatory diseases are characterised by abnormal accumulation ofinflammatory cells including monocytes/macrophages, granulocytes, plasmacells, lymphocytes and platelets. Along with tissue endothelial cellsand fibroblasts, these inflammatory cells release a complex array oflipids, growth factors, cytokines and destructive enzymes that causelocal tissue damage.

One form of inflammatory response is neutrophilic inflammation which ischaracterized by infiltration of the inflamed tissue by neutrophilpolymorphonuclear leucocytes (PMN), which are a major component of hostdefence. Tissue infection by extracellular bacteria represents theprototype of this inflammatory response. On the other hand, variousnon-infectious diseases are characterized by extravascular recruitmentof neutrophils. This group of inflammatory diseases includes chronicobstructive pulmonary disease, adult respiratory distress syndrome, sometypes of immune-complex alveolitis, cystic fibrosis, bronchitis,bronchiectasis, emphysema, glomerulonephritis, active phases ofrheumatoid arthritis, gouty arthritis, ulcerative colitis, certaindermatoses such as psoriasis and vasculitis. In these conditionsneutrophils are thought to play a crucial role in the development oftissue injury which, when persistent, can lead to the irreversibledestruction of the normal tissue architecture with consequent organdysfunction. Thereby tissue damage is mainly caused by the activation ofneutrophils followed by their release of proteinases and increasedproduction of oxygen species.

Chronic obstructive pulmonary disease (COPD) is basically a conditiondescribed by the progressive development of airflow limitation that isnot fully reversible (ATC, 1995). Most patients with COPD have threepathological conditions: bronchitis, emphysema and mucus plugging. Thisdisease is characterised by a slowly progressive and irreversibledecrease in forced expiratory volume in the first second of expiration(FEV₁), with relative preservation of forced vital capacity (FVC)(Barnes, N. Engl. J. Med. (2000), 343(4): 269-280). In both asthma andCOPD there is significant, but distinct, remodelling of airways. Most ofthe airflow obstruction is due to two major components,alveolar_destruction (emphysema) and small airways obstruction (chronicobstructive bronchitis). In COPD it is mainly characterised by profoundmucus cell hyperplasia.

Cigarette smoking, air pollution and other environmental factors aremajor causes of the disease. The causal mechanism remains currentlyundefined but oxidant-antioxidant disturbances are strongly implicatedin the development of the disease. COPD is a chronic inflammatoryprocess that differs markedly from that seen in asthma, with differentinflammatory cells, mediators, inflammatory effects and responses totreatment (Keatings et al., Am. J. Respir. Crit. Care Med. (1996), 153:530-534). Primarily, neutrophil infiltration of the patient's lungs is acharacteristic of this disease.

Elevated levels of proinflammatory cytokines like TNF-α, and especiallychemokines like IL-8 and GRO-α seem to play a very important role inpathogenesis of this disease. Platelet thromboxane synthesis is alsoenhanced in patients with COPD (Keatings et al., Am. J. Respir. Crit.Care Med. (1996), 153: 530-534; Stockley and Hill, Thorax (2000), 55(7):629-630). Most of the tissue damage is caused by activation ofneutrophils followed by their release of (metallo)proteinases, andincreased production of oxygen species (Repine et al., Am. J. Respir.Crit. Care Med. (1997), 156: 341-357; Barnes, Chest (2000), 117(2Suppl): 10S-14S).

Most therapeutic endeavour is directed towards the control of symptoms(Barnes, Trends Pharm. Sci. (1998), 19(10): 415-423; Barnes, Am. J.Respir. Crit. Care Med. (1999) 160: 572-S79; Hansel et al., Expert Opin.Investig. Drugs (2000) 9(1): 3-23). Symptoms usually equate with airflowlimitation and bronchodilators are the therapy of choice.

Prevention and treatment of complications, prevention of deteriorationand improved quality and length of life are also primary goals stated inthe three key international guidelines for the management of COPD(Culpitt and Rogers, Exp. Opin. Pharmacother. (2000) 1(5): 1007-1020;Hay, Curr. Opin. Chem. Biol. (2000), 4: 412-419). Basically, most of thecurrent therapeutic research has been focused on mediators involved inthe recruitment and activation of neutrophils, or attenuation ofconsequences of their undesirable activation (Stockley et al., Chest(2000), 117(2 Suppl): 58S-62S).

There are a number of reports on immunomodulatory action of macrolideantibiotics in vitro (Labro, J. Antimicrob. Chemother. (1998), 41 (SupplB): 37-46; Labro, Clin. Microb. Rev. (2000), 13(4): 615-650; Wales andWoodhead, Thorax (1999), 54 (Suppl 2): S58-S62). Macrolide antibioticsare macrocyclic compounds containing for example a 12-, 14-, 16- or17-membered lactone ring and 1 to 3 sugar residues, which are linked toeach other or to the aglucone by glycosidic bounds. Known members ofmacrolide antibiotics are for example carbomycin, erythromycin,leucomycin and spiramycin.

The most important findings with regard to macrolide interaction withphagocytic inflammatory cells in vitro concern the inhibitory effects onoxidant production by stimulated cells (Labro et al., J. Antimicrob.Chemother. (1989), 24 (4): 561-572; Umeki, Chest (1993), 104: 1191-1193;Wenisch at al., Antimicrob. Agents Chemother. (1996), 40(9): 2039-2042)and modulation of pro-inflammatory and anti-inflammatory cytokinerelease by these cells (Labro et al., J. Antimicrob. Chemother. (1989),24 (4): 561-572; Khan et al, Internat. J. Antimicrob. Agents. (1999),11: 121-132; Morikawa et al., Antimicrob. Agents and Chemother. (1996),40(6): 1366-1370; Sugiyama et al., Eur. Respir. J. (1999), 14:1113-1116). In addition, several macrolides directly stimulateexocytosis (degranulation) by human neutrophils in vitro (Abdelghaffaeet al., Antimicrob. Agents Chemother. (1994), 38(7): 1548-1554; Vazifehet al., Antimicrob. Agents Chemother. (1998), 42 (8): 1944-1951). In theexperimental inflammatory model of carrageenin pleurisy in the rat, somemacrolide antibiotics like roxithromycin, clarithromycin anderythromycin, but not azithromycin, were found to show anti-inflammatoryactivity which probably depended on their ability to prevent theproduction of pro-inflammatory mediators and cytokines. In this model ofacute inflammation, NO production, TNF-α levels or PGE₂ weresignificantly reduced by the antibiotic pre-treatment (Ianario et al.,J. Pharmacol. Exp. Ther. (2000), 292: 156-163).

Erythromycin administration also caused anti-inflammatory effects inzymosan-induced peritonitis in rats (Agen et al., Agents Actions (1993),38(1-2): 85-90). Roxithromycin was reported to be active in reducing theacute inflammatory reaction through a mechanism different fromconventional anti-inflammatory agents such as indomethacin. In anotherstudy, roxithromycin was demonstrated to be effective in a standardanimal model used for evaluating the effects of anti-inflammatory drugson carrageenin-induced paw oedema, whereas clarithromycin andazithromycin showed modest activity (Scaglione and Rossini, J.Antimicrob. Chemother. (1998), 41, Suppl B: 47-50).

Some macrolide antibiotics, like erythromycin, clarithromycin androxithromycin have already been used as anti-inflammatory drugs,especially for the treatment of diffuse panbronchiolitis. Reports on theuse of macrolides for diseases like rheumatoid arthritis and cysticfibrosis are available (Arayssi et al., Programm and Abstracts of the4^(th) International conference on macrolides, azalides, streptograminsand ketolides, 21-23 Jan. 1998, Barcelona, Spain, Abstract 6; Singh, J.Assoc. Phys. India (1989), 37: 547; Jaffe et al., Lancet (1998), 351:420). With regard to relevant pharmacological effects of macrolides, ithas been reported that erythromycin inhibits hypersecretion due toinhibition of mucus and water secretion from epithelial cells. It alsoinhibits neutrophil accumulation in the inflammatory region due toinhibition of their attachment to the capillary vessels, IL-8 secretionfrom the epithelial cells and secretion of IL-8 and LTB₄ from theneutrophil, itself. Its beneficial effects in diffuse panbronchiolitisalso include a reduction of superoxide production, and reduction of theproteolytic enzyme levels in lungs.

Azithromycin has been shown to significantly improve lung function, butthe underlying mechanism was unclear (Jaffe et al., Lancet (1998), 351:420), while roxithromycin was reported to suppress the growth of nasalpolyp fibroblasts (Nonaka et al., Am. J. Rhinol. (1999), 13: 267-272,Yamada et al., Am. J. Rhinol. (2000), 14: 143-148).

While strong evidence in published literature exists that macrolideswith a 14-membered ring such as erythromycin, clarithromycin androxithromycin inhibit in vitro IL-8 production and neutrophilchemotaxis, evidence even in vitro is limited that macrolides with a15-membered ring such as azithromycin exert a similar anti-inflammatoryaction (Criqui et al., Eur. Respir. J. (2000), 15: 856-862).

In U.S. Pat. No. 4,886,792 inhibitory effects on neutrophildegranulation of 15-membered macrolactones were described, but theselacked the sugar substituents of azithromycin. Azithromycin has beenreported to induce apoptosis in human neutrophils in vitro, but waswithout effect on oxidative metabolism or IL-8 production (Koch et al.,J. Antimicrob. Chemother. (2000), 46: 19-26). Only one study has shownazithromycin to inhibit neutrophil chemotaxis and active oxygengeneration in vitro (Sugihara, Kansenshogaku Zasshi J. Jpn. Assoc.Infec. Dis. (1997), 71: 329-336). Also, azithromycin has been shown notto change TNFα, IL-1β or IL-6 levels of alveolar macrophages or blood(Aubert et al., Pul. Pharmacol. Ther. (1998), 11: 263-269).

The possibility that azithromycin, by virtue of its 15-membered ring,lacks the requisite structure conferring anti-inflammatory activity tothe 14-membered macrolides has been suggested and is made more likely bythe observation that 16-membered macrolides such as josamycin do notreduce IL-8 production (Takizawa et al., Am. J. Resp. Crit.Care Med.(1997), 156: 266-271; Criqui et al., Eur. Respir. J. (2000), 15:856-862).

In comparison with macrolide antibiotics having a 14-membered ringmacrolide compounds with a 15-membered ring possess several advantages.For example erythromycin whose structure is characterised by a14-membered aglucone ring is in acidic medium easily converted intoanhydroerythromycin, which is an inactive C-6/C-12 metabolite of aspiroketal structure (Kurath et al., Experienta (1971), 27: 362). Incontrast to its parent antibiotic erythromycin azithromycin exhibits animproved stability in acidic medium. Furthermore, azithromycin exhibitsa significantly higher concentration in tissues. Due to its improved invitro activity against gram-negative microorganisms there was eventested the possibility of a one-day dose (Ratshema et al., Antimicrob.Agents Chemother. (1987), 31: 1939).

Thus, the technical problem underlying the present invention is toprovide improved means, in particular improved processes andapplications useful for the therapy of neutrophil-dominatednon-infective inflammatory diseases, in which the active ingredientexhibits the advantageous anti-inflammatory activities of macrolidecompounds having a 14-membered lactone ring as well as the improvedstability and high tissue concentration of macrolide compounds having a15-membered ring.

The present invention solves the above problem by the use of an activeingredient selected from the group consisting of azithromycin, apharmaceutically acceptable derivate thereof, a pharmaceuticallyacceptable hydrate thereof, a pharmaceutically acceptable complex orchelate thereof and a pharmaceutically acceptable salt thereof, for theproduction of pharmaceutical compositions for the treatment ofneutrophil-dominated, non-infective inflammatory diseases in humanbeings and animals.

In contrast to the limited effects of azithromycin on neutrophilfunction in vitro described in the art according to the presentinvention it has been surprisingly found that azithromycin administeredto humans in vivo has a broad range of anti-inflammatory activities andis highly useful in the therapy of inflammatory diseases characterizedby neutrophil infiltration and neutrophil associated tissue damage.

In a trial conducted on healthy volunteers the influence of azithromycinon selected inflammation-relevant parameters was followed up. Thereby itwas found that the administration of azithromycin stimulates thedegranulation of human neutrophils as shown by a strong change of theconcentration of primary azurophilic granular enzymes, such asmyeloperoxidase (MPO), N-acetyl-β-D-glucosaminidase (NAGA) andβ-glucuronidase.

The biological relevance of MPO activity in granulocytes is a strongoxygen-dependent antimicrobial activity connected to mobilisation of allgranules in the inflammatory granulocytes in the inflammation process,especially after phagocytic stimulus by immune complexes. Afterazithromycin application MPO activities in blood smear neutrophilsstrongly decreased and returned to baseline only after 28 days. Therebyit was found that degranulation presented with lower MPO neutrophildensity as determined with cytochemistry was associated with lower MPOELISA concentrations in neutrophil lysates.

N-acetyl-β-D-glucosaminidase (NAGA) and β-glucuronidase are lyosomalenzymes, both of which are located in azurophilic (primary orperoxidase-positive) granules of neutrophils. Since during inflammationdegranulation of neutrophils occurs, both enzymes are markers ofdegranulation and can be used for estimation of neutrophil reactivity.The studies on azithromycin showed that after azithromycin applicationthe activity of NAGA in serum increased considerably. Even 28 days afterthe last azithromycin dose serum NAGA values were still 70% higher thaninitial values. The increase in NAGA in serum was accompanied by adecrease in enzyme activity in PMN. The activity of β-glucuronidase inserum did not show any changes during the first day after the lastazithromycin dose but afterwards increased. 28 days after the last doseof azithromycin the activity of β-glucuronidase was 40% higher thaninitially. Activities of β-glucuronidase in PMN decreased within thenext hours after the last azithromycin dose but than increased. 28 daysafter the last azithromycin dose β-glucuronidase activity in PMN wasmuch higher than initially.

Furthermore, according to the invention it was shown that azithromycininhibits the generation of reactive oxygen species from stimulatedneutrophils as demonstrated by the inhibition of chemiluminescencegenerated from stimulated neutrophils. That azithromycin is an inhibitorof neutrophil oxidative burst was further demonstrated by using acytochrom c assay system. The studies also revealed that azithromycinhas also a long-term effect on the concentration of cellular gluthationeperoxidase (GSHPx) and gluthatione reductase, two enzymes that controlthe biological effects of free radicals which have been implicated inthe pathogenesis of a large number of diseases. Free radical productionand disturbance in redox status can modulate the expression of a varietyof inflammatory molecules, affecting certain cellular processes leadingto inflammatory processes. Thus azithromycin provides a basis for thetreatment of a variety of diseases such as COPD in which neutrophilradical production becomes excessive.

The studies also confirmed that azithromycin induces apoptosis, i.e. theprogrammed cell death, of certain cell types. Apoptosis is an importantmechanism to complete an immune response. A three-day administration ofazithromycin exerted a delayed pro-apoptotic effect on granulocytes, asindicated by the morphology of blood smear. The number of apoptoticcells reached its maximum 28 days after the last azithromycin dosesuggesting a decreased number of active, potentially damagingneutrophils.

In the study other anti-inflammatory effects of azithromycin were alsodetected. In contrast to previous studies (Koch et al., J. Antimicrob.Chemother. (2000), 46: 19-26) it was found according to the inventionthat azithromycin has a marked inhibitory effect on the release of IL-8and also GRO-α. Interleukin-8 (IL-8) is a member of theneutrophil-specific CXC subfamiliy of chemokines. It is a potentneutrophil chemotactic and activating factor (Oppenheim, Ann. Rev.Immunol. (1999), 9: 617). IL-8 is expressed in response to inflammatorystimuli. IL-8 delays spontaneous and TNF-α-mediated apoptosis of humanneutrophils. In contrast to the effect on IL-8, azithromycin increasesgradually the serum concentration of the cytokine IL-1, whereby thehighest IL-1 concentration was found 24 h after the last azithromycindose. However, the serum concentration of another cytokine, IL-6, wascontinuously decreased.

In contrast to earlier reports (Semaan et al., J. Cardiovasc. Pharmacol.(2000), 36: 533-537) in which azithromycin treatment did notsignificantly affect the plasma levels of soluble VCAM, studiesconducted according to the present invention clearly showed a markeddecrease of plasma levels of sVCAM already 24 h after azithromycintreatment.

The results obtained according to the invention demonstrate that athree-day treatment of healthy human subjects, with a standardantibacterial dosage regimen of azithromycin, exerts acute effects onneutrophil granular enzymes, oxidative burst, oxidative protectivemechanisms and neutrophil chemokines and circulating IL-1, IL-6, IL-8,as well as delayed effects on neutrophil apoptosis and soluble adhesionmolecules.

According to the present invention, therefore, azithromycin can be usedas a valuable prophylactic and/or therapeutic agent inneutrophil-dominated, non-infective inflammatory diseases.

The following definitions are set forth to illustrate and define themeaning and scope of the various terms used to describe the presentinvention.

The term “neutrophil-dominated non-infective inflammatory disease”refers to inflammatory diseases, disorders or conditions which resultfrom tissue damage, chemical irradiation or immune processes, but notfrom the invasion of microorganisms such as viruses, bacteria, fungi,protozoa or the like, und which are characterised by infiltration of theinflamed tissue by neutrophils which are the first inflammatory cells toenter the tissue and to amplify the inflammatory response. In some ofnon-infective inflammatory diseases neutrophils remain the dominant celltype within the inflamed area, even when the response is prolongedbecause of the continued presence of stimuli for neutrophil infiltrationand activation. Examples therefore are chronic obstructive pulmonarydisease (COPD), adult respiratory distress syndrome (ARDS) andneutrophilic dermatoses. Other neutrophil-dominated non-infectiveinflammatory diseases include diseases which have an underlying stimulusto the chronicity of the pathology, which is not dependent onneutrophils. For example autoimmune diseases are mainly due to thedevelopment of immune responses to normal structural components of thebody and involve activation of T lymphocytes, with the possibleproduction of autoantibodies by B lymphocytes. In rheumatoid arthritis(RA), for example, immune reactions are directed against structuralcomponents of the joints. However, in RA and other autoimmune diseasesacute flare-ups occur, which are characterised by intense neutrophilinfiltration and activation. These active phases of chronic autoimmuneinflammation are neutrophil-dominated, for instance resulting inpronounced accumulation of neutrophils in the synovial fluids ofpatients with RA. In some autoimmune diseases, the generation ofautoantibodies is pronounced, leading to deposition in the tissue ofimmune complexes of antigen and autoantibody and activation of thecomplement system. Neutrophils enter the tissue in an attempt to engulfthe immune complexes and the neutrophil infiltration and activation isexacerbated by activated complement factors. An example of this type ofdisease is a renal disease, in particular glomerulonephritis resultingin pronounced kidney damage.

Therefore, the term “neutrophil-dominated non-infective inflammatorydisease” includes, without being restricted to, chronic obstructivepulmonary disease (COPD), adult respiratory distress symptome (ARDS),bronchitis, bronchiectasis, emphysema, cystic fibrosis, inflammatorybowel disease, gouty arthritis, autoimmune diseases characterised byacute neutrophil-dominated phases, such as rheumatoid arthritis,autoimmune diseases, in which neutrophil infiltration is exacerbated byactivated complement factors, such as glomerulonephritis, and skindiseases, in particular all kinds of neutrophilic dermatoses includingpsoriasisform dermatoses, such as psoriasis and Reiter's syndrome,autoimmune bullous dermatoses, vessel-based neutrophilic dermatoses suchas leukocytoclastic vasculitis, Sweet's syndrome, pustular vasculitis,erythema nodosum and familial Mediterranean fever, and pyodermagangrenosum.

The term “neutrophil-dominated non-infective inflammatory disease”includes also all accompanying diseases, disorders or conditions whichoccur as a result of a neutrophil-dominated non-infective inflammatorydisease and which can affect tissues or organs of the body other thanthat affected by the inflammatory disease itself. An example thereforeare extraintestinal diseases such as uveitis and chronic hepatitis whichcan result from inflammatory bowel disease.

The term “active ingredient” or “active agent” refers to any substanceswhich can affect or recognise biological cells or parts thereof, inparticular cell organelles or cellular components. Such activeingredients or agents are of a chemical nature. In particular, suchactive ingredients or agents are diagnostics or therapeutics. In thecontext of the present invention the term “active ingredients” or“active agents” refers in particular to therapeutics, i.e. substances,which can be administered as a preventive measure or during the courseof a disease, disorder or condition to organisms in need of such atreatment in order to prevent or to reduce or to abolish a disease,disorder or condition, in particular a neutrophil-dominatednon-infective inflammatory disease.

In the context of the present invention, the term “treatment” refers toa prophylactic and/or therapeutic effect of a drug or medicament whichin turn is defined as a pharmaceutical composition comprising apharmaceutically or diagnostically effective compound in combinationwith at least one additive, such as a carrier.

“Azithromycin” refers to the macrolide compoundN-methyl-11-aza-10-deoxo-10-dihydroerythromycin A(9-deoxo-9-dihydro-9a-methyl-9a-aza-9a-homoerythromycin A) with a15-membered azalactone ring which can be obtained by the Beckmannrearrangement of erythromycin A-oxime followed by Eschweiler-Clarkereductive N-methylation essentially as described in U.S. Pat. No.4,517,359, U.S. Pat. No. 4,328,334 and BE 892,357, whereby thedisclosure contents of these documents with regard to the methods forproduction of azithromycin are completely incorporated in the disclosurecontent of the present application.

The term “pharmaceutically acceptable derivative thereof” refers tonon-toxic functional equivalents or derivatives of azithromycin, whichcan be obtained by substitution of atoms or molecular groups or bonds ofthe azithromycin molecule, whereby the basic structure of azithromycinis not changed, and which differ from the azithromycin structure in atleast one position. The term “pharmaceutically acceptable derivative”includes for example O-methyl derivatives of azithromycin which can beobtained essentially as described in U.S. Pat. No. 5,250,518, wherebythe disclosure content of this document with regard to the methods forproduction of O-methyl derivatives is completely incorporated in thedisclosure content of the present application.

The term “pharmaceutically acceptable derivative” includes also estersof azithromycin which retain, upon hydrolysis of the ester bond, thebiological effectiveness and properties of azithromycin and are notbiologically or otherwise undesirable. Techniques for the preparation ofpharmaceutically acceptable esters are for instance disclosed in MarchAdvanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New York (1985)p. 1152. Pharmaceutically acceptable esters useful as prodrugs aredisclosed in Bundgaard, H., ed., (1985) Design of Prodrugs, ElsevierScience Publishers, Amsterdam.

The term “pharmaceutically acceptable hydrate thereof” refers tonon-toxic solid or fluid compounds of azithromycin retaining thebiological activities of azithromaycin and generated by the process ofhydration whereby one or more molecules of water associate with theazithromycin molecule due to dipole forces. The term includes forexample mono- and dihydrates of azithromycin.

The term “pharmaceutically acceptable salts” refers to the non-toxicalkali metal, alkaline earth metal, and ammonium salts commonly usedincluding the ammonium, barium, calcium, lithium, magnesium, potassium,protamine zinc salts and sodium, which are prepared by methods known inthe art. The term also includes non-toxic; i.e. pharmaceuticallyacceptable acid addition salts, which are generally prepared by reactingazithromycin with a suitable organic or inorganic acid, such as acetate,benzoate, bisulfate, borate, citrate, fumarate, hydrobromide,hydrochloride, lactate, laurate, maleate, napsylate, oleate, oxalate,phosphate, succinate, sulfate, tartrate, tosylate, valerate, etc.

The term “pharmaceutically acceptable acid addition salt” refers tosalts which retain the biological effectiveness and properties of thefree bases and which are not biologically or otherwise undesirable,formed with inorganic acids such as hydrobromic acid, hydrochloric acid,nitric acid, phosphoric acid, sulfuric acid, and organic acids such asacetic acid, benzoic acid, cinnamic acid, citric acid, ethanesulfonicacid, fumaric acid, glycolic acid, maleic acid, malic acid, malonicacid, mandelic acid, menthanesulfonic acid, oxalic propionic acid,p-toluenesulfonic acid, pyruvic acid, salicylic acid, succinic acid,tartaric acid, etc.

The salts of the invention can be obtained by dissolving azithromycin inan aqueous or aqueous/alcoholic solvent or in other suitable solventswith an appropriate base and then isolating the obtained salt of theinvention by evaporating the solution, by freezing and lyophilization orby addition of another solvent, e.g. diethylether, to the aqueous and/oralcoholic solution of the azithromycin salt including the separation ofunsoluble crude salt. For the preparation of alkali azithromycin salts,alkali metal carbonates or hydrogencarbonates are preferably used. Theprepared salts are freely soluble in water.

The term “pharmaceutically acceptable complex or chelate thereof” refersto non-toxic complexes and chelates of azithromycin with bivalent and/ortrivalent metals which can be obtained essentially as described in U.S.Pat. No. 5,498,699, whereby the disclosure content of this document withregard to the methods for production of complexes and chelates ofazithromycin is completely incorporated in the disclosure content of thepresent application. As complex- and chelate-forming metals, metals ofthe II and III group which can form physiologically tolerated compounds,in particular Mg²⁺, Al³⁺, Fe³⁺, Rh³⁺, La³⁺ and Bi³⁺ can be used.Preferably the ratio of azithromycin to metal is in the range of 1:1 to1:4. In order to obtain complexes and chelates of azithromycin theantibiotic is reacted in form of a free base or salt, in particular as ahydrochloride, with a salt of a bivalent and/or trivalent metal in aratio of 2:1 at ambient temperature in an aqueous solution or in amixture of water/alcohol at a pH of 8.0 to 11.0 with a metal hydroxideand/or carbonate, subsalicylate or a gel thereof. Preferred examplesinclude chelates of azithromycin with antacids chosen from the group ofsalts of Al, Mg and Bi, chelates of azithromycin with sucralfate andchelates of azithromycin with bismuth-subsalicylate which are in theform of a gel.

The term “pharmaceutically or therapeutically acceptable carrier” refersto a carrier medium which does not interfere with the effectiveness ofthe biological activity of the active ingredients and which is not toxicto the host or patient.

The active ingredient selected from the group consisting ofazithromycin, a pharmaceutically acceptable derivate thereof, apharmaceutically acceptable hydrate thereof, a pharmaceuticallyacceptable complex or chelate thereof and a pharmaceutically acceptablesalt can also be administered to animals, including mammals such asrodents and primates, including humans, to prevent or to reduce or toabolish neutrophil-dominated non-infective inflammatory diseases. Thus,the present invention encompasses methods for therapeutic treatment ofsuch disorders or diseases that comprise administering an activeingredient of the invention in amounts sufficient to reach the desiredeffect of azithromycin in vivo. For example, the active agent oringredient of the present invention can be administered in atherapeutically or pharmaceutically effective amount to treat a varietyof non-infective inflammatory diseases, including but not limited toCOPD, ARDS and neutrophilic dermatoses.

“Therapeutically or pharmaceutically effective amount” as applied toazithromycin or the azithromycin containing compounds and compositionsof the present invention refers to the amount of a compound orcomposition sufficient to induce a desired biological result. Thatresult can be alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. In thepresent invention, the result will for instance in a particularlypreferred embodiment involve preventing, abolishing and/or reducing thesymptoms or causes of a neutrophil-dominated non-infective inflammatorycondition by acute effects on neutrophil granular enzymes, oxidativeburst, oxidative protective mechanisms and neutrophil chemokines andcirculating IL-1, IL-6, IL-8, as well as delayed effects on neutrophilapoptosis and soluble adhesion molecules. In a preferred embodiment theactive ingredients of the present invention will be administeredprophylactically prior to the outbreak of a neutrophil-dominatednon-infective inflammatory disease.

Accordingly, the present invention also provides pharmaceuticalcompositions comprising, as an active ingredient azithromycin, apharmaceutically acceptable derivate thereof, a pharmaceuticallyacceptable hydrate thereof, a pharmaceutically acceptable complex orchelate thereof and a pharmaceutically acceptable salt in associationwith a pharmaceutical carrier or diluent. The compositions of thisinvention can be administered systematically or topically, in particularby intravascular oral, pulmonary, parenteral, e.g. intramuscular,intraperitoneal, intravenous (IV) or subcutaneous injection orinhalation, e.g. via a fine powder formulation, transdermal, nasal,vaginal, rectal, or sublingual routes of administration and can beformulated in dosage forms appropriate for each route of administration.The active agent or ingredient is adminstered preferably in apharmaceutically effective amount.

Solid dosage forms for oral administration include capsules,lingualettes, tablets, pills, powders, liposomes, patches, time delayedcoatings and granules. In such solid dosage forms, the active compoundis admixed with at least one inert pharmaceutically acceptable carriersuch as lactose, sucrose, or starch. Such dosage forms can also compriseadditional substances other than inert diluents, e.g., lubricatingagents such as magnesium stearate. In the case of capsules, tablets, andpills, the dosage forms may also comprise bulking and/or buffering aswell as flavouring agents. Tablets and pills can additionally beprepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, with the elixirscontaining inert diluents commonly used in the art, such as water.Besides such inert diluents, compositions can also include adjuvants,such as salts for varying the osmotic pressure, pH-adjusting compounds,skin penetration agents, wetting agents, emulsifying and suspendingagents, and sweetening, flavouring, and perfuming agents.

Pharmaceutical compositions according to the present invention forparenteral administration include sterile aqueous or non-aqueoussolutions, suspensions, or emulsions. Examples of non-aqueous solventsor vehicles are propylene glycol, polyethylene glycol, vegetable oils,such as olive oil and corn oil, gelatine, and injectable organic esterssuch as ethyl oleate. Such dosage forms may also contain adjuvants suchas preserving, wetting, emulsifying, and dispersing agents. They may besterilised by, for example, filtration through a bacteria retainingfilter, by incorporating sterilising agents into the compositions, byirradiating the compositions, or by heating the compositions. They canalso be manufactured using sterile water, or some other sterileinjectable medium, immediately before use.

Formulations for injection will comprise a physiologically-acceptablemedium, such as water, saline, PBS, aqueous ethanol, aqueous ethyleneglycols and the like. Water soluble preservatives which may be employedinclude sodium bisulfite, sodium thiosulfate, ascorbate, benzalkoniumchloride, chlorobutanol, thimerosal, phenylmercuric borate, parabens,benzyl alcohol and phenylethanol. These agents may be present inindividual amounts of from about 0.001 to about 5% by weight andpreferably about 0.01 to about 2%. Suitable water soluble bufferingagents that may be employed are alkali or alkaline earth carbonates,phosphates, bicarbonates, citrates, borates, acetates, succinates andthe like, such as sodium phosphate, citrate, borate, acetate,bicarbonate and carbonate. Additives such as carbomethylcellulose may beused as a carrier in amounts of from about 0.01 to about 5% by weight.The formulation will vary depending upon the purpose of the formulation,the particular mode employed for treating a disease, the intendedtreatment, etc.

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as cocoa butter or a suppository wax. Compositions fornasal or sublingual administration are also prepared with standardexcipients well known in the art.

The compositions containing the active agent or ingredient of thepresent invention can be administered for prophylactic and/ortherapeutic treatments. In therapeutic applications, compositions areadministered to a patient already suffering from a disease, as describedabove, in an amount sufficient to cure or at least partially arrest thesymptoms of the disease and its complications, i.e. a therapeuticallyeffective amount.

In prophylactic applications, compositions containing the active agentor ingredient of the present invention are administered to a patientsusceptible to or otherwise at risk of a particular disease. Such anamount is defined to be a “prophylactically effective dose.” In thisuse, the precise amounts again depend upon the patient's state of healthand weight.

The pharmaceutical compositions of the present invention may also beadministered in the form of a depot, such as a slow release composition.Such a slow release composition may include particles of the activeagent or ingredient in a matrix, made e.g. from collagen.

The quantities of the active agent or ingredient necessary for effectivetherapy will depend upon many different factors, including means ofadministration, target site, physiological state of the patient, andother medicants administered.

The active agent or ingredient of the present invention selected fromthe group consisting of azithromycin, a pharmaceutically acceptablederivate thereof, a pharmaceutically acceptable hydrate thereof, apharmaceutically acceptable complex or chelate thereof and apharmaceutically acceptable salt thereof are effective in treatingneutrophil-dominated non-infective inflammatory diseases whenadministered in a range of from about 10 mg to about 2000 mg per day, inparticular from about 30 to about 1500 mg. The specific dose employed isregulated by the particular condition being treated, the route ofadministration, as well as by the judgement of the attending cliniciandepending upon factors such as the severity of the condition, and theage and general condition of the patient.

The active agents or ingredients of the present invention may beadministered alone or together with other medicaments currently used forthe treatment of neutrophil-dominated non-infective inflammatorydiseases such as non-steroidal anti-inflammatory agents, such as methylxanthine non-steroidal anti-inflammatory agents, steroidalanti-inflammatory agents, immunomodulating agents, immunosuppressiveagents, bronchodilating agents, antirheumatic agents, corticosteroids,β2-agonists, cholinergic antagonists, and the like, whereby the dose ofthe latter can possibly be reduced by 50% or 25% due to theanti-inflammatory effects of the active ingredients of the presentinvention.

The composition, preferably the water-soluble composition, of theinvention may further contain a water-soluble protein injectable intobody fluids without showing any substantial pharmacological activity atthe concentration used in one unit dosage form of the present invention(hereinafter, “water-soluble protein”). As such a water-soluble protein,serum albumin, globulin, collagen and/or gelatine are preferred. Thisprotein can be added in an amount generally employed in injectablepharmaceutical compositions. Thus, for example, the weight ratio betweenthe water-soluble protein and the active agent or ingredient of thepresent invention is about 0.0001:1 to 100:1, preferably about 0.001:1to about 10:1 or more preferably about 0.01:1 to about 1:1.

Continuing, the invention also relates to the aforementioned activeagents or ingredients themselves and compositions containing them, inparticular, in dried and/or pure form or in an aqueous oraqueous/alcoholic solution. The pH of a solution prepared from thewater-soluble composition or an active agent of the present inventionshould be such that said pH will not exert any adverse influence uponthe activity of the pharmacologically active peptide, but is within anacceptable range for injections in general and further, such that saidpH will neither cause a great change in viscosity of the solution norallow formation of a precipitate or the like. Thus the solution shouldpreferably have a pH of about 4 to 7, preferably 5 to 6, in particular5.3 to 5.5.

When the water-soluble composition of the invention is converted into anaqueous solution for administration, the concentration of thepharmacologically active agent or ingredient or salt thereof in saidsolution should preferably be about 0.0000001 to 10% (w/v), morepreferably about 0.000001 to 5% (w/v) or most preferably about 0.00001to 1% (w/v).

The composition of the present invention should preferably have a unitdosage form containing the pharmacologically active agent or ingredientof the invention and, if necessary, together with further additives suchas the above mentioned water-soluble protein. Thus, for example, the twoor three components mentioned above are made to occur in an ampule orvial by dissolving or suspending them in sterile water or sterilephysiological saline. In this case, the method of preparation maycomprise admixing a solution of the pharmacologically active agent oringredient and further, if necessary, a solution of the additive oradding the additive in a powder form to a solution of thepharmacologically active agent or ingredient or any other combination ofadequate procedures. The dosage form may also be prepared by addingsterile water or sterile physiological saline to a lyophilizate orvacuum-dried powder in which the pharmacologically active agent, and ifnecessary the additive, coexist. This unit dosage form may contain oneor more conventional additives such as pH adjusting agents (e.g.glycine, hydrochloric acid, sodium hydroxide), local anesthetics (e.g.xylocaine hydrochloride, chlorobutanol), isotonizing agents (e.g. sodiumchloride, mannitol, sorbitol), emulsifiers, adsorption inhibitors (e.g.Tween® 60 or 80), talcum, starch, lactose and tragacanth, magnesiumstearate, glycerol, propylen glycol, preserving agents, benzyl alcohol,methylhydroxy benzoate and/or oleum arachid hydrogen. This unit dosageform may further contain pharmaceutically acceptable excipients such aspolyethylene glycol 400 or dextran.

The composition of the present invention is made by admixing theseingredients according to a conventional method. The goal of admixing theingredients of the present composition should be such that the activityof the pharmacologically active agent is maintained and bubble formationminimised during the process. The ingredients are put into a vessel (forexample a bottle or drum) either at the same time or in any order. Theatmosphere in the vessel can be, for example, sterile clean air orsterile clean nitrogen gas. The resultant solution can be transferred tosmall vials or ampules and can be further subjected to lyophilization.

The liquid form or the lyophilizate powder form of the composition ofthe present invention may be dissolved or dispersed in a solution of abiodegradable polymer such as poly(lactic-glycolic) acid copolymer,poly(hydroxybutyric acid), poly(hydroxybutyric-glycolic) acid copolymer,or the mixture of these, and then may be formulated, for example, tofilms, microcapsules (microspheres), or nanocapsules (nanospheres),particularly in the form of soft or hard capsules.

In addition, the composition of the present invention encapsulated inliposomes comprising phospholipids, cholesterol or the derivatives ofthese can be further dispersed in physiological saline or a hyaluronicacid solution dissolved in physiological saline.

The soft capsule may be filled with the liquid form of the compositionof the present invention. The hard capsule may be filled with thelyophilizate powder of the composition of the present invention, or thelyophilizate powder of the present composition may be compressed totablets for rectal administration or oral administration respectively.

Of course, the composition of the present invention can be supplied in apre-filled syringe for self-administration.

Although only preferred embodiments of the invention are specificallydescribed above, it will be appreciated that modifications andvariations of the invention are possible without departing from thespirit and intended scope of the invention. Further preferredembodiments of the present invention are listed in the claims.

EXAMPLE

A trial on healthy volunteers was conducted and the influence ofazithromycin given in a dosage of 3×500 mg on selectedinflammation-relevant parameters was followed up.

Drug Administration, Blood Sampling and Plasma

Each subject received two standard 250 mg capsules of azithromycin(Sumamed®, PLIVA Zagreb) on three consecutive days. Immediately beforethe treatment and 2 h and 30 min, 24 h and 28 days after the third andlast dose of azithromycin blood was collected from the cubital vein intoEDTA-containing tubes. Aliquots were taken for cell counting, smearpreparation, polymorphonuclear cell and serum isolation.

Analysis of Primary Azurophilic Granular Enzymes

Leucocyte granules are membrane-bound organells containing an array ofantimicrobial proteins. Apart from containing degradative enzymes thatmay be extracellulary secreted from the neutrophil or else dischargedinto phagocytic vesicles, the membranes of many types of these granulesand vesicles contain important molecules such as certain receptors (e.g.fMLP receptor) and cytochrome b of NADPH oxidase.

a) Analysis of Myeloperoxidase

The enzyme myeloperoxidase (MPO) is a 135,000 dalton protein containingtwo heavy and two light chains of 55,000 and 15,000 daltons. MPO issituated in primary or azurophil granules of granulocytic cells. Thefunction of MPO is to provide reactive oxygen metabolites that areessential for microbicidal activity of neutrophils. The generation ofoxygen metabolites is dependent on components of MPO-negative granules(which harbour the flavocytochrome b₅₅₈, an essential component of theNADPH oxidase) and on components of azurophil MPO positive granules. MPOtransforms the relatively innocuous product of the NADPH oxidase, H₂0₂,to hypochlorous acid. The biological relevance of MPO activity ingranulocytes is a strong oxygen-dependent antimicrobial activityconnected to mobilisation of all granules in the inflammatorygranulocytes in the inflammation process, especially after phagocyticstimulus by immune complexes.

The activity of MPO was assessed from the intensity of staining ofneutrophils in blood smears and in cell lysates by ELISA. After fixationin ethanol-formaldehyde, smears were incubated in a substrate solutioncontaining hydrogen peroxide and benzidine (SIGMA). After incubation,smears were counterstained with Giemsa solution. MPO value positivity_of100 granulocytes was evaluated and scored from 0 to 4+ on the basis ofthe intensity of the precipitated dye in cytoplasm. Therefore, the valueof the score could be from 0 to 400. Normal values of score range(290-390) were taken from this study before azithromycin administration.MPO activity was also evaluated on the digital image of smear taken witha digital camera under the high magnification (×1000) of lightmicroscope. MPO activities in blood smear neutrophils decreased from 2 hand 30 min to 24 h after the last azithromycin dose and returned tobaseline after 28 days (Table 1). The concentration of MPO enzymeprotein determined by ELISA in lysates of neutrophils is shown inTable 1. The change in neutrophil enzyme protein followed the samepattern as that in intracellular enzyme activity, decreasing from 2 hand 30 min to 24 h after the last dose of azithromycin and returning tobaseline after 28 days. Both methodological approaches of MPOdetermination confirmed each other. Degranulation presented with lowerMPO neutrophil density determined_with cytochemistry was associated withlower MPO ELISA concentrations in neutrophil lysates.

b) Analysis of N-acetyl-β-D-glucosaminidase (NAGA) and β-glucuronidase

Glycosidases are enzymes that catalyse hydrolysis of glycosidic bonds ofoligosacharides and other glycosides. They are specific to theglycosidic part of substrate molecule. N-acetyl-β-D-glucosaminidase(NAGA) and β-glucuronidase are such enzymes. They are lysosomal enzymes,both located in azurophilic (primary; peroxidase-positive) granules ofneutrophils. Since degranulation of neutrophils is present duringinflammation, many authors choose these enzymes as markers ofdegranulation and for estimation of neutrophil reactivity. The catalyticconcentration of both enzymes in serum and in neutrophil lysates wasdetermined using the fluorimetric method described by O'Brien et al.(New Engl. J. Med. (1970) 283: 15-20) for NAGA and Glaser & Sly (J. Lab.Clin. Med. (1973) 82: 969) for β-glucuronidase.

The results showed (Table 1) that activity of NAGA in serum increasedabout 30% 2 h and 30 min after the last dose. 24 hours after the lastdose it was approximately 50% higher than the initial values. 28 dayslater, serum NAGA values were still 70% higher than initial values. Theincrease in NAGA in serum was accompanied by a decrease in enzymeactivity in PMN. 2 h and 30 min after the last dose a decrease of about70% in NAGA in granulocytes was determined. 24 hours later, NAGAactivity in PMN increased by about 30% but it was still about 40% lowercompared to initial values. After 28 days the activity of NAGA increased40% over the initial values (Table 1).

The activity of β-glucuronidase in serum did not show any changes duringthe first 24 hours after the last dose. 28 days later serum values wereabout 40% higher than initially. Activities of β-glucuronidase in PMNdecreased by about 20% after 2 h and 30 min and by about 50% 24 hourslater compared to initial values. However 28 days later, β-glucuronidaseactivity in PMN was much higher (about 300%) compared to the initialvalues (Table 1).

When analysing activities of glycosidases it is obvious thatazithromycin in healthy volunteers induced release of 40-50% enzymesfrom azurophilic granules within 24 hours after the last dose. Thedecrease of NAGA activity in PMNs was accompanied by an increase inserum. Serum activities of the two enzymes showed a slight increase overbaseline (before azithromycin) 2 h and 30 min and 24 h after the lastdose of the drug, increasing a further 28 days later (Table 1).

In contrast, activities of the two enzymes in neutrophil lysatesdecreased in the hours after the last dose of azithromycin, the fall inNAGA activity being maximal after 2 h and 30 min and returning tobaseline after 28 days. The cellular activity of β-glucuronidase wasstill falling 24 h after the last dose of azithromycin and increased towell above baseline levels after 28 days (Table 1).

In summary, enzymes released from neutrophil primary azurophilicgranules tended to be present in serum at slightly higher activities 2 hand 30 min to 24 h after azithromycin administration, while over thesame time period, their activities were lower in peripheral bloodneutrophils, suggesting that they were being released by degranulation.NAGA was released early after azithromycin, while MPO andβ-glucuronidase exhibited a delayed release. Recovery of these enzymeactivities also varied.

Studies on Neutrophil Oxidative Burst

All aerobic organisms use oxygen for the production of energy. However,there are many indications that the advantages of using oxygen areassociated with a risk that the oxidative process may also cause injury.During phagocytosis when neutrophils are stimulated, they undergo anoxidative burst, with generation and release of reactive oxygenmetabolites. These reactive oxygen species serve as the major mechanismby which phagocytes mediate their antimicrobial effect. The reactionsare characterised by rapid oxygen uptake followed by reduction of oxygento superoxide (O₂ ⁻). This is catalysed by NADPH oxidase using NADPH orNADH as electron donor. When these defence mechanisms are directedinappropriately, tissue damage occurs.

a) Determination of Chemiluminescence Generation

The generation of reactive oxygen species by activated cells isfrequently determined by the measurement of chemiluminescence (CL). Theradical species formed react with a photon-producing chemical (e.g.Luminol) and the resulting light emission is measured with a photocell.Chemiluminescence is detectable as a result of the stimulation (e.g.fMLP) of leucocytes and is a measure of their oxidative cytotoxicactivity (Allen et al., Biochem. Biophys. Res. Commun. (1972), 47: 679).

The results of the study presented in Table 1 show, that azithromycininhibits chemiluminescence generated from stimulated neutrophilsisolated from the blood of humans treated with azithromycin.

b) Cytochrome C Assay System

Neutrophils were incubated with cytochrome c and stimulated with fMLP(Cohen and Chovaniec, 1978, J. Clin. Invest. 61: 1081-1087). Absorbancesat 550 nm and 540 nm were recorded and the results were expressed asdelta A.

The oxidative burst of neutrophils in response to the bacterial peptidefMLP was inhibited by the 3 day dosing with azithromycin (Table 1).Using both cytochrome c and luminol as assay systems, inhibition wasalready detectable 2 h and 30 min after the last dose of azithromycin,was greater after 24 h and had not returned to normal 28 days later.

Consequently, azithromycin is to be considered as an inhibitor of theoxidative burst. Thus, azithromycin provides a basis for a variety ofdiseases in which neutrophil radical production (oxidative burst)becomes excessive such as COPD.

Analysis of Glutathione Peroxidase and Glutathione Reductase

Oxygen free radicals and lipid peroxides have been implicated in thepathogenesis of a large number of diseases. The biological effects offree radicals are controlled in vivo by a wide range of antioxidantssuch as α-tocopherol (vitamin E), ascorbic acid (vitamin C), β-carotene,reduced glutathione (GSH) and antioxidant enzymes (superoxide dismutase,SOD, glutathione peroxidase GSHPx, catalase, CAT) (Benabdeslam et al.,Clin. Chem. Lab. Med. (1999), 37: 511-516; Mates et al., Blood CellsMol. (1999), 25: 103-109). Recently, antioxidant functions have beendefinitively linked to anti-inflammatory and/or immunosuppressiveproperties (Mates et al., Blood Cells Mol. (1999), 25: 103-109). Freeradical production and disturbance in redox status can modulate theexpression of a variety of inflammatory molecules (Sundaresan et al.,Science (1995), 270: 296-299; Kaouass et al., Endocrine (1997), 6:187-194), affecting certain cellular processes leading to inflammatoryprocesses, both exacerbating inflammation and effecting tissue damage(Tsai et al., FEBS Lett. (1997), 436: 411-414).

Cellular glutathione peroxidase (GSHPx) is a tetrameric protein in whicheach of the four identical subunits contains one atom of selenium (Se)in the form of selenocysteine at the active site (Misso et al., J.Leukoc. Biol. (1998), 63: 124-130). GSHPx plays a role in H₂0₂detoxification and converts lipid hydroperoxides to nontoxic alcohols(Akkus et al., Clin. Chim. Acta (1996), 244: 221-227); Urban et al.,Biomed & Pharmacother. (1997), 51: 388-390). In this study, in healthyvolunteers treated with azithromycin alterations in the PMNintracellular GSHPx activity were determined using the commerciallyavailable kit RANSEL (Randox Laboratories). GSHPx catalyses theoxidation of glutathione by cumene hydroperoxide. In the presence ofglutathione reductase and NADPH the oxidised glutathione is immediatelyconverted to the reduced form with a concomitant oxidation of NADPH toNADP⁺. The decrease in absorbance at 340 nm is measured.

Glutathione reductase is an ubiquitous enzyme that catalyses thereduction of oxidised glutathione (GSSG) to glutathione (GSH).Glutathione reductase is essential for the glutathione redox cycle thatmaintains adequate levels of reduced cellular GSH. GSH serves as anantioxidant, reacting with free radicals and organic peroxides, in aminoacid transport, and as a substrate for the GSHPx and glutathioneS-transferases in the detoxification of organic peroxides and metabolismof xenobiotics. Glutathione reductase was determined using theBIOXYTECH® GR-340™ colorimetric assay for glutathione reductase (OXISInternational, Inc.). Briefly, oxidation of NADPH to NADP⁺ is catalysedby a limiting concentration of glutathione reductase.

GSHPx activity in neutrophil lysates (expressed per number of cells) wasunchanged 2 h and 30 min after the last dose of azithromycin, butdecreased significantly 24 h after this last dose (Table 1). Theactivity had returned to baseline 28 days later. Glutathione reductaseactivity in cell lysates (expressed per number of cells) showed asimilar tendency, decreasing significantly 2 and 30 min and 24 h afterthe last dose of azithromycin, returning to normal values and thenreaching higher levels than normal 28 days after the treatment (Table1).

Analysis of Apoptosis

Three-day administration of azithromycin exerted a delayed pro-apoptoticeffect on granulocytes, as indicated by morphology of blood smears. Theresults are presented in Table 1. The number of apoptotic cells countedincreased continuously after the three day dosing with azithromycin,achieving statistical significance 28 days after the last dose. Anincreased number of apoptotic cells suggest a decreased number ofactive, potentially damaging neutrophils.

Analysis of Cytokines and Chemokines

Other acute, but potentially anti-inflammatory effects of azithromycinwere also detected in this study.

Interleukin-8, a member of the neutrophil-specific CXC subfamily ofchemokines is a potent neutrophil chemotactic and activating factor(Oppenheim, J.J. Ann. Rev. Immunol. (1999), 9: 617). It binds to atleast two G protein-coupled receptors (IL-8R1 and IL-8R2). Thesereceptors are functionally different. Responses, such as cytososlic freeCa²⁺ changes and release of the granule enzymes, are mediated throughboth receptors, whereas the respiratory burst and the activation ofphospholipase D depend exclusively on stimulation through IL-8R1 (Johneset al., Proc. Natl. Acad. Sci. USA (1996), 93: 6682-6686). IL-8 is a keymediator in the recruitment of circulating neutrophils. This chemokineis expressed in response to inflammatory stimuli, and is secreted by avariety of cell types, including lymphocytes, epithelial cells,keratinocytes, fibroblasts, endothelial cells, smooth muscle cells andneutrophiles. In the latter instance, IL-8 is one of the most abundantlysecreted (and most extensively) studied cytokines produced byneutrophils. Interestingly enough, neutrophils represent the primarycellular target for IL-8, to which they respond by chemotaxis, releaseof granule content, respiratory burst, up-regulation of cell surfacereceptors, increased adherence to non-stimulated endothelial cells, andtransmigration across the endothelium. Agents capable of stimulating theproduction of IL-8 by human neutrophils are: TNF-α, IL-1β, GM-CSF,leukotriene B₄, PAF, fMLP, lactoferrin, LPs and many others (Cassatella,M. A., Adv. Immunol. (1999), 73: 369-509). IL-8 delays spontaneous andTNF-α-mediated apoptosis of human neutrophils. (Kettritz et al., KidneyInt. (1998), 53: 84-91). IL-8 is the pre-dominant C—X—C chemokine andthe dominant neutrophil chemoattractant accumulating in supernatant ofLPS-stimulated human alveolar macrophages (Goodmann et al., Am. J.Physiol. (1998), 275: L87-L95).

Erythromycin was reported to have an inhibitory effect on IL-8expression in human epithelial cells and this mode of action is probablyof relevance for its clinical effectiveness (Takizawa et al., Am. J.Respir. Crit. Care Med. (1997), 156: 266-271).

Roxithromycin is also capable of reducing IL-8 production in nasal polypfibroblasts (Nonaka et al., Acta Otolaryngol. (1998) Suppl. 539: 71-75).In synoviocytes from rheumatoid arthritis, the production of IL-1α,IL-6, IL-8, GM-CSF could be inhibited by chlarithromycin (Matsuoka etal., Clin. Exp. Immunol. (1996), 104(3): 501-8). Ex vivo assessment ofIL-8 production in whole blood also confirmed the potential oferythromycin for inhibiting IL-8 production (Schultz et al., J.Antimicrob. Chemother. (2000), 46: 235-240. A similar finding hasrecently been reported for human bronchial epithelial cells (Desaki M.et al., Biochim. Biophys Res. Commun. (2000) 267: 124-128). A recentstudy, however, reported a lack of azithromycin modulatory effect onIL-8 production of PMN in vitro (Koch et al., J. Antimicrob. Chemother.(2000), 46: 19-26).

Cytokine and chemokine concentrations were determined using ELISA kits.Several different response patterns were seen in serum cytokine andchemokine concentrations following three-day administration ofazithromycin. Rapid and pronounced decreases in the plasmaconcentrations of the neutrophil-stimulating chemokine, IL-8, and GR0-αwere observed 2 h and 30 min and 24 h after the last dose ofazithromycin (Table 1. The concentration of IL-8 returned essentially tobaseline after 28 days, while that of GRO-□ was decreased at this time.

These data clearly demonstrate the acute inhibitory effect ofazithromycin on the release of IL-8 ex vivo, extending this propertyalso to inhibition of the release of the chemokine GRO-□. It should bestated, however, that the serum chemokine concentration was measured.Therefore one cannot draw any conclusion as to the cellular source(s) ofthe chemokines.

The low baseline serum concentration of IL-1 gradually increased afterthe last dose of azithromycin, achieving statistical significance after24 h (Table 1). The concentration had returned to baseline 28 days afterazithromycin. In contrast, the serum concentration of IL-6 exhibited acontinuous decrease, achieving statistical significance 28 days afterthe last dose of azithromycin (Table 1).

Analysis of Adhesion Molecules

In contrast to earlier reported data (Semaan et al., J. Cardiovasc.Pharmacol. (2000), 36: 533-537) in which azithromycin treatment did notsignificantly affect the plasma levels of soluble VCAM, in this study adecrease in serum sVCAM was observed 24 h after the last dose ofazithromycin, remaining significantly reduced after 28 days, indicatingthat azithromycin has the potential to inhibit both the generation ofneutrophil chemotactic peptides and the expression and release ofadhesion molecules for activated leucocytes (Table 1). For quantitativedetermination of serum concentration of human sVCAM, an ELISA kit wasused (R&D systems, UK)

Proteins in PMN samples were determined according to the method ofBradford (Anal. Biochem. (1976) 72: 248-254) using bovine serum albuminas a standard. TABLE 1 UNITS baseline 2 h and 30 min 24 hours 28 daysDEGRANULATION myeloperoxidase (score) 337 ± 29  326 ± 26  315 ± 22* 347± 18  myeloperoxidase (density) 105 ± 13  130 ± 16* 131 ± 17* 115 ± 19 myeloperoxidase (PMN) μg/mg protein  54.22 ± 12.61 70.85 ± 19.91 26.74 ±2.51* 70.01 ± 17.62 NAGA (PMN) nmol × 10⁻⁶ cells × min⁻¹ 4.15 ± 01.6 1.13 ± 0.72* 2.62 ± 1.6* 5.95 ± 3.7  β-glucuronidase (PMN) nmol ×10^(−6 cells × min) ⁻¹ 4.12 ± 2.7  3.21 ± 2.3  1.58 ± 0.4* 15.37 ± 11.4*NAGA (serum) μmol × L⁻¹ × min⁻¹ 9.16 ± 1.6  11.52 ± 2.2  13.7 ± 1.5*14.87 ± 1.9*  β-glucuronidase (serum) μmol × L⁻¹ × min⁻¹ 2.88 ± 0.7 3.01 ± 0.6  2.95 ± 0.5  3.93 ± 1.2* CYTOKINES (serum) IL-1 pg/mL 0.291 ±0.11  0.533 ± 0.15   1.07 ± 0.19* 0.29 ± 0.20 IL-6 pg/mL  3.4 ± 1.05 2.7 ± 1.49  2.5 ± 1.48  1.15 ± 0.61* CHEMOKINES (serum) IL-8 pg/mL29.47 ± 15.44 10.61 ± 3.81*  14.60 ± 10.75* 23.03 ± 19.72 GRO-α pg/mL124.1 ± 33.02  109.6 ± 30.35*  107.9 ± 27.83*  90.4 ± 22.32* APOPTOSIS(WBC) apoptopic cells/1000 WBC 0.333 ± 0.655 0.833 ± 1.029 1.417 ± 1.2402.583 ± 2.02* ADHESION MOLECULES sV-CAM ng/mL 13.59 ± 2.90  12.21 ±4.12  10.29 ± 2.12* 10.74 ± 2.05* OXIDATIVE BURST (PMN) fMLP-luminolA.U. 29335 ± 1957  14774 ± 1175*  5053 ± 3804*  9879 ± 13880*fMLP-cytochrom c ΔA 0.020 ± 0,014  0,007 ± 0.015* −0,018 ± 0.010*−0,0011 ± 0,0010* GLUTATHION PEROXIDASE (PMN) mU/10⁶PMN 5.3 ± 2.0 5.3 ±2.9  1.6 ± 1.3* 8.0 ± 5.2 GLUTATHION REDUCTASE (PMN) mU/10⁶PMN 9.63 ±1.16  7.39 ± 1.23*  7.91 ± 0.87* 11.27 ± 2.24**p < 0.01 vs baseline (Wilcoxon).

1. A method for treating neutrophil-dominated, non-infectiveinflammatory diseases in human beings and animals comprisingadministering to said human beings and animals a therapeutically orpharmaceutically effective amount of an active ingredient whichcomprises azithromycin, or a pharmaceutically acceptable derivative,hydrate, complex, chelate, or salt thereof.
 2. The method according toclaim 1, whereby the active ingredient is an 0-methyl-derivative ofazithromycin.
 3. The method according to claim 1, whereby the activeingredient is an ester of azithromycin.
 4. The method according to claim1, whereby the active ingredient is a monohydrate of azithromycin. 5.The method according to claim 1, whereby the active ingredient is adihydrate of azithromycin.
 6. The method according to claim 1, wherebythe active ingredient is a complex or chelate of azithromycin with metalions.
 7. The method according to claim 6, whereby the ratio betweenazithromycin to metal is 1:1 to 1:4.
 8. The method according to claim 6,whereby the metal ions are bivalent metal ions.
 9. The method accordingto claim 6, whereby the metal ions are trivalent metal ions.
 10. Themethod according to claim 1, whereby the active ingredient is an alkalimetal, alkaline earth metal, or an ammonium salt of azithromycin. 11.The method according to claim 1, whereby the active ingredient is anacid addition salt of azithromycin.
 12. The method according to claim11, whereby the acid addition salt is formed with an inorganic acid. 13.The method according to claim 11, whereby the inorganic acid ishydrobromic acid, nitric acid, phosphoric acid or sulphuric acid. 14.The method according to claim 11, whereby the acid addition salt isformed with an organic acid.
 15. The method according to claim 14,whereby the organic acid is acetic acid, benzoic acid, cinnamic acid,citric acid, ethanesulfonic acid, fumaric acid, glycolic acid, maleicacid, malic acid, malonic acid, mandelic acid, methanesulfonic acid,oxalic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid,succinic acid or tartaric acid.
 16. The method according to claim 1,whereby the pharmaceutical compositions contain the active ingredient inan amount sufficient to abolish or to reduce the disease or to stop itsprogression.
 17. The method according to claim 16, whereby thepharmaceutical compositions are administered one to three times a day ina dose of 10 mg to 200 mg active ingredient.
 18. The method according toclaim 17, whereby the pharmaceutical compositions are administered oneto three times a day in a dose of 30 mg to 1500 mg active ingredient.19. The method according to claim 1, whereby the pharmaceuticalcompositions are orally administered in solid or liquid dosage forms.20. The method according to claim 19, whereby the solid pharmaceuticalcompositions for oral administration are capsules, lingualettes,tablets, pills, powders, liposomes, patches, time delayed coatings andgranules.
 21. The method according to claim 19, whereby the solidpharmaceutical compositions for oral administration. contain at leastone inert pharmaceutically acceptable carrier.
 22. The method accordingto claim 21, whereby the inert pharmaceutical carrier is lactose,sucrose, or starch.
 23. The method according to claim 19, whereby thesolid pharmaceutical compositions for oral administration compriseadditional substances selected from the group consisting of lubricatingagents such as magnesium stearate, bulking and/or buffering agents andflavouring agents.
 24. The method according to claim 19, whereby thesolid pharmaceutical compositions for oral administration are preparedwith enteric coatings:
 25. The method according to claim 19, whereby theliquid pharmaceutical compositions for oral administration arepharmaceutically acceptable emulsions, solutions, suspensions or syrups.26. The method according to claim 25, whereby the liquid pharmaceuticalcomposition for oral administration contains at least one inertpharmaceutical carrier.
 27. The method according to claim 26, wherebythe inert pharmaceutical carrier is water or physiological saline. 28.The method according to claim 25, whereby the liquid pharmaceuticalcomposition for oral administration comprises additional substances,selected from the group consisting of adjuvants, salts for varying theosmotic pressure, pH-adjusting compounds, skin penetration agents,wetting agents, emulsifying and suspending agents.
 29. The methodaccording claim 1, whereby the pharmaceutical compositions areparenterally administered.
 30. The method according to claim 29, wherebythe pharmaceutical compositions for parenteral administration areinfusions or injections.
 31. The method according to claim 29, wherebythe pharmaceutical compositions for parenteral administration aresterile aqueous or non-aqueous solutions, suspensions or emulsions. 32.The method according to claim 29, whereby the pharmaceuticalcompositions for parenteral administration comprise non-aqueous solventsor vehicles.
 33. The method according to claim 32, whereby thenon-aqueous solvents or vehicles are propylene glycol, polyethyleneglycol, vegetable oils, such as olive oil and corn oil, gelatine, andinjectable organic esters such as ethyl oleate.
 34. The method accordingto claim 29, whereby the pharmaceutical compositions for parenteraladministration comprise adjuvants such as preserving, wetting,emulsifying, and dispersing agents.
 35. The method according to claim 1,whereby the pharmaceutical compositions are rectally or vaginallyadministered.
 36. The method according to claim 35, whereby thepharmaceutical compositions for rectal or vaginal administration aresuppositories, clysters or foams.
 37. The method according to claim 35,whereby the pharmaceutical compositions for rectal or vaginaladministration contain excipients such as cocoa butter or a suppositorywax.
 38. The method according to claim 1, whereby the pharmaceuticalcompositions for the treatment of neutrophil-dominated, non-infectiveinflammatory diseases contain one or more additional active ingredientsuseful for the treatment of such diseases selected from the groupconsisting of non-steroidal anti-inflammatory agents, steroidalanti-inflammatory agents, bronchodilating agents, antirheumatic agents,immunomodulating agents, immunosuppressive agents, corticosteroids,β2-agonists and cholinergic antagonists.
 39. The method according toclaim 38, whereby the, dose of the additional active ingredients isreduced in comparison to pharmaceutical compositions, containingexclusively one of the additional active ingredients.
 40. Apharmaceutical composition for the treatment of neutrophil-dominated,non-infective inflammatory diseases in human beings and animalscomprising an active ingredient which comprises azithromycin, or apharmaceutically acceptable derivate, hydrate, complex, chelate, or saltthereof
 41. The pharmaceutical composition according to claim 40,whereby the active ingredient is an O-methyl-derivative or an ester ofazithromycin.
 42. The pharmaceutical composition according to claim 40,whereby the active ingredient is a monohydrate or a dehydrate ofazithromycin.
 43. The pharmaceutical composition according to claim 40,whereby the active ingredient is a complex or chelate of azithromycinwith bivalent or trivalent metal ions.
 44. The pharmaceuticalcomposition according to claim 43, whereby the ratio betweenazithromycin and metal ions is 1:1 to 1:4.
 45. The pharmaceuticalcomposition according to claim 40, whereby the active ingredient is analkali metal, alkaline earth metal, or an ammonium salt of azithromycin.46. The pharmaceutical composition according to claim 40, whereby theactive ingredient is an acid addition salt of azithromycin.
 47. Thepharmaceutical composition according to claim 46, whereby the acidaddition salt is formed with an inorganic acid, such as hydrobromicacid, nitric acid, phosphoric acid or sulphuric acid.
 48. Thepharmaceutical composition according to claim 46, whereby the acidaddition salt is farmed with an organic acid, such as acetic acid,benzoic acid, cinnamic acid, citric acid, ethanesulfonic acid, fumaricacid, glycolic acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid; oxalic acid, p-toluenesulfonic acid, pyruvicacid, salicylic acid, succinic acid or tartaric acid.
 49. Thepharmaceutical composition according to claim 40, whereby the activeingredient is contained in an amount sufficient to abolish or to reducethe disease or to stop its progression.
 50. The pharmaceuticalcomposition according to claim 40, comprising one or more additionalactive ingredients useful for the treatment of such diseases selectedfrom the group consisting of non-steroidal anti-inflammatory agents,steroidal anti-inflammatory agents, bronchodilating agents,antirheumatic agents, immunomodulating agents, immunosuppressive agents,corticosteroids, β2-agonists and cholinergic antagonists.
 51. Thepharmaceutical composition according to claim 50, whereby the dose ofthe additional active ingredients is reduced in comparison topharmaceutical compositions, containing exclusively one of theadditional active ingredients.
 52. A method for the production of apharmaceutical composition comprising azithromycin, or apharmaceutically acceptable derivative, hydrate, chelate, or saltthereof as an active ingredient, for the treatment ofneutrophil-dominated, non-infective inflammatory diseases in humanbeings and animals which comprises (i) admixing the active ingredientwith additives and optionally with additional active ingredients usefulfor the treatment of such diseases, (ii) dissolving or suspending theresulting admixture in sterile aqueous or aqueous/alcoholic solution,(iii) adjusting the pH of the solution to a value of about 4 to 7 by theuse of pH adjusting agents, and (iv) filling the pH adjusted solutioninto vials or ampules.
 53. The method according to claim 52, whereby theadditional active ingredients are selected from the group consisting ofnon-steroidal anti-inflammatory agents, steroidal anti-inflammatoryagents, bronchodilating agents, antirheumatic agents, immunomodulatingagents, immunosuppressive agents, corticosteroids, β2-agonists andcholinergic antagonists.
 54. The method of claim 1, wherein theneutrophil-dominated, non-infective inflammatory disease is selectedfrom the group consisting of an adult respiratory distress syndrome(ARDS), emphysema, a neutrophil dermatosis, auto-immune bullousdermatoses, vessel-based neutrophilic dermatoses, an autoimmune diseasein which neutrophil infiltration is exacerbated by activated complementfactors, and an auto-immune disease characterized by acuteneutrophil-dominated phases.
 55. The method of claim 54, wherein theneutrophil dermatosis is selected from the group consisting of psoriasisand Reiter's syndrome.
 56. The method of claim 54, wherein thevessel-based neutrophilic dermatoses disease is selected from the groupconsisting of leukocytoclastic vasculitis, Sweet's syndrome, pustularvasculitis, erythemanodosum, familial Mediterranean fever, and pyodermagangrenosum.
 57. The method of claim 54, wherein the auto-immune diseasein which neutrophil infiltration is exacerbated by activated complementfactors, is a renal disease.
 58. The method of claim 57, wherein therenal disease is glomerulonephritis.
 59. The method of claim 54, whereinthe auto-immune disease characterized by acute neutrophil-dominatedphases is rheumatoid arthritis.
 60. The pharmaceutical composition ofclaim 40, wherein the neutrophil-dominated, non-infective inflammatorydisease is selected from the group consisting of adult respiratorydistress syndrome (ARDS), emphysema, a neutrophil dermatosis,auto-immune bullous dermatoses, vessel-based neutrophilic dermatoses, anauto-immune disease in which neutrophil infiltration is exacerbated byactivated complement factors, and an autoimmune disease characterized byacute neutrophil-dominated phases.
 61. The pharmaceutical composition ofclaim 60, wherein the neutrophil dermatosis is selected from a groupconsisting of psoriasis and Reiter's syndrome.
 62. The pharmaceuticalcomposition of claim 60, wherein the vessel-based neutrophilicdermatoses is selected from the group consisting of leukocytoclasticvasculitis, Sweet's syndrome, pustular vasculitis, erythema nodosum,familial Mediterranean fever, and pyoderma gangrenosum.
 63. Thepharmaceutical composition of claim 60, wherein the auto-immune diseasein which neutrophil infiltration is exacerbated by activated complementfactors, is a renal disease.
 64. The pharmaceutical composition of claim63, wherein the renal disease is glomerulonephritis.
 65. Thepharmaceutical composition of claim 60, wherein the autoimmune diseasecharacterized by acute neutrophil-dominated phases is rheumatoidarthritis.
 66. The method of claim 52, wherein the neutrophil-dominated,non-infective inflammatory disease is selected from the group consistingof adult respiratory distress syndrome (ARDS), emphysema, a neutrophildermatosis, auto-immune bullous dermatoses, vessel-based neutrophilicdermatoses, an auto-immune disease in which neutrophil infiltration isexacerbated by activated complement factors, and an autoimmune diseasecharacterized by acute neutrophil-dominated phases.
 67. The method ofclaim 66, wherein the neutrophil dermatosis is selected from the groupconsisting of psoriasis and Reiter's syndrome.
 68. The method of claim66, wherein the vessel-based neutrophilic dermatoses is selected fromthe group consisting of leukocytoclastic vasculitis, Sweet's syndrome,pustular vasculitis, erythema nodosum, familial Mediterranean fever, andpyoderma gangrenosum.
 69. The method of claim 66, wherein theauto-immune disease in which neutrophil infiltration is exacerbated byactivated complement factors, is a renal disease.
 70. The method ofclaim 69, wherein the renal disease is glomerulonephritis.
 71. Themethod of claim 66, wherein the autoimmune disease characterized byacute neutrophil-dominated phases is rheumatoid arthritis.